Normal automaticity in sinoatrial node cells (SANC) involves intracellular Ca2+ cycling within a coupled-clock system: periodic local, subsarcolemmal Ca2+ releases (LCRs) from sarcoplasmic reticulum (Ca2+ clock) activate an inward Na+-Ca2+ exchange current that accelerates the diastolic depolarization prompting the ensemble of surface membrane ion channels (membrane clock) to generate the next action potential (AP). Whether intracellular Ca2+ regulates SANC AP firing rate on a beat-to-beat basis is controversial. We loaded single isolated SANC with a caged Ca2+ buffer, NP-EGTA, and simultaneously recorded membrane potential and intracellular Ca2+. Prior to introduction of the caged Ca2+ buffer, spontaneous LCRs during diastolic depolarization (DD) were tightly coupled to rhythmic APs (r2=0.9). The buffer markedly prolonged the decay time (T50) of the AP-induced Ca2+ transient and partially depleted the SR load level, suppressed spontaneous diastolic LCRs and uncoupled them from AP generation, and caused AP firing to become markedly slow and dysrhythmic. When Ca2+ was acutely released from the caged compound by flash photolysis, intracellular Ca2+ dynamics were acutely restored and rhythmic APs resumed immediately at a normal rate. After a few rhythmic cycles, however, these effects of the flash waned as interference with Ca2+ dynamics by the caged buffer was reestablished. Our results directly support the hypothesis that a system of an intracellular Ca2+ clock coupled to a surface membrane voltage clock regulates normal SANC automaticity on a beat-to-beat basis. We investigated recently whether a change in beating rate (i.e., due to autonomic receptor stimulation) is accompanied by rhythm variation (beat-to-beat variability in rate). Autonomic receptor stimulations affected both the beating rate and rhythm variability in isolated rabbit pacemaker cells; i.e. decrease in beating rate occurred concurrently with increase in rhythm variability. Under physiological conditions, application of low concentrations of caffeine (2-4mM) to isolated single rabbit SANC acutely reduces their spontaneous action potential cycle length (CL) and increases Ca2+ transient amplitude for several cycles. Numerical simulations, using a modified Maltsev-Lakatta coupled-clock model, faithfully reproduced these effects, and also the effects of CL prolongation and dys-rhythmic spontaneous beating produced by cytosolic Ca2+ buffering and an acute CL reduction produced by flash-induced Ca2+ release from a caged Ca2+ buffer, that we had reported previously. Three contemporary numerical models (including the original Maltsev-Lakatta model) failed to reproduce the experimental results. In our new model, Ca2+ releases acutely change the CL via activation of the Na+/Ca2+ exchanger current. Time-dependent cycle length reductions after flash-induced Ca2+ releases (memory effect) are linked to changes in Ca2+ available for pumping into sarcoplasmic reticulum which, in turn, changes the sarcoplasmic reticulum Ca2+ load, diastolic Ca2+ releases and Na+/Ca2+ exchanger current. These results support the idea that Ca2+ regulates CL in cardiac pacemaker cells on a beat-to-beat basis, and suggest a more realistic numerical mechanism of this regulation. To unravel clock-crosstalk effects on changes in CL on a beat-to-beat basis we desynchronized clock function by directly inhibiting either the M or Ca2+ clock, confirmed that the other clock was not directly inhibited and measured average CL and LCR period, and their changes on a beat-to-beat basis. To inhibit the M clock, we employed a range of concentrations of ivabradine (IVA), an If inhibitor, and to inhibit the Ca2+ clock we employed a range of concentrations of cyclopiazonic acid (CPA), a SR Ca2+ pump inhibitor. Our results revealed that direct perturbation of only the M or Ca2+ clock indirectly impacts the other clock to desynchronize clock function, which results in not only prolongation of the LCR period but also in an increase in its beat-to-beat variability. Therefore, our results provide new evidence for a general theory of normal pacemaker cell rate and rhythm.